1. Field of Invention
The present invention relates to a mass spectrometer in general and in particular to a mass spectrometer that combines the use of time-of-flight mass spectrometry and ion-trap mass spectrometry in a single mass spectrometer.
2. Description of Related Art
Mass spectrometers are instruments that are used to determine the chemical composition of substances and the structures of molecules. In general they consist of an ion source where neutral molecules are ionized, a mass analyzer where ions are separated according to their mass/charge ratio, and a detector. Mass analyzers come in a variety of types, including magnetic field (B) instruments, combined electrical and magnetic field or double-focusing instruments (EB or BE), quadrupole electric field (Q) instruments, and time-of-flight (TOF) instruments. In addition, two or more analyzers may be combined in a single instrument to produce tandem (MS/MS) mass spectrometers. These include triple analyzers (EBE), four sector mass spectrometers (EBEB or BEEB), triple quadrupoles (QqQ) and hybrids (such as the EBqQ).
In tandem mass spectrometers, the first mass analyzer is generally used to select a precursor ion from among the ions normally observed in a mass spectrum. Fragmentation is then induced in a region located between the mass analyzers, and the second mass analyzer is used to provide a mass spectrum of the product ions. Tandem mass spectrometers may be utilized for ion structure studies by establishing the relationship between a series of molecular and fragment precursor ions and their products. Alternatively, they are now commonly used to determine the structures of biological molecules in complex mixtures that are not completely fractionated by chromatographic methods. These may include mixtures of, for example, peptides, glycopeptides or glycolipids. In the case of peptides, fragmentation produces information on the amino acid sequence.
One type of mass spectrometers is time-of-flight (TOF) mass spectrometers. The simplest version of a time-of-flight mass spectrometer, illustrated in FIG. 1 (Cotter, Robert J., Time-of-Flight Mass Spectrometry: Instrumentation and Applications in Biological Research, American Chemical Society, Washington, D.C., 1997), the entire contents of which is hereby incorporated by reference, consists of a short source region 10, a longer field-free drift region 12 and a detector 14. Ions are formed and accelerated to their final kinetic energies in the short source region 10 by an electric field defined by voltages on a backing plate 16 and drawout grid 18. Other grids or lenses 17 may be added to the source region to enhance extraction and to improve the mass resolution by reducing the initial velocity distribution. The longer field-free drift region 12 is bounded by drawout grid 18 and an exit grid 20.
In the most common configuration, the drawout grid 18 and exit grid 20 (and therefore the entire drift length) are at ground potential, the voltage on the backing plate 16 is V, and the ions are accelerated in the source region to an energy: mv2/2=z eV, where m is the mass of the ion, v is its velocity, e is the charge on an electron, and z is the charge number of the ion. The ions then pass through the drift region 12 and their (approximate) flight time(s) is given by the formula:t=[(m/z)/2 eV]1/2D  (I)which shows a square root dependence upon mass. Typically, the length 1 of source region 10 is of the order of 0.5 cm, while drift lengths (D) ranges from 15 cm to 8 meters. Accelerating voltages (V) can range from a few hundred volts to 30 kV, and flight time are of the order of 5 to 100 microseconds. Generally, the accelerating voltage is selected to be relatively high in order to minimize the effects on mass resolution arising from initial kinetic energies and to enable the detection of large ions. For example, the accelerating voltage of 20 KV (as illustrated for example in FIG. 1) has been found to be sufficient for detection of masses in excess of 300 kDaltons (kDa).
Mass resolution can be improved by pulsing one or more of the source elements such as the backing plate 16 or the grid 17. Other times-dependent pulses or waveforms may also be applied to the source (Kovtoun, S. V., English, R. D. and Cotter, R. J., Mass Correlated Acceleration in a Reflectron MALDI TOF Mass Spectrometer: An Approach for enhanced Resolution over a Broad Range, J. Amer. Soc. Mass Spectrom. 13 (2002) 135–143).
Mass resolution may also be improved by the addition of a reflectron (Mamyrin, B. A., Karataev, V. I., Shmikk, D. V. Zagulin, V. A. Sov. Phys. JET 37 (1973) 45). A conventional reflectron is essentially a retarding electrical field which decelerates the ions to zero velocity, and allows them to turn around and return along the same or nearly the same path. Ions with higher kinetic energy (velocity) penetrate the reflectron more deeply than those with lower kinetic energy, and thus have a longer path to the detector. Ions retain their initial kinetic energy distributions as they reach the detector; however, ions of different masses will arrive at different times.
An example of a time-of-flight mass spectrometer utilizing a reflectron is shown schematically in FIG. 2 (same numerals in FIG. 1 and FIG. 2 are used to indicate same elements however positioned differently). The reflectron may be single stage or dual-stage. In both single-stage and dual-stage reflectrons, a stack of electrodes 32 (also called ion lenses), each connected resistively to one another, provide constant retarding field regions that are separated by one grid 34 in the single stage reflectron 30. In the most common case, grids and lenses are constructed using ring electrodes. In the case of grid 34 illustrated in FIG. 2, the ring electrode is covered with a thin wire mesh.
In single-stage reflectrons, a single retarding region is used as and (approximate) ion flight times are given by the formula:t=[(m/z)/2 eV]1/2 [L1+L2+4d]  (II)which has the same square-root dependence expressed in Equation (I). The terms, in addition to those expressed in Equation (I), are L1, L2 and d. L1 and L2 are the lengths of the linear drift regions illustrated in FIG. 2, respectively, in the forward and return directions, and d is the average penetration depth.
While reflectrons were originally intended to improve mass resolution for ions formed in an ion source region, they have more recently been exploited for recording the mass spectra of product ions formed outside the source by metastable decay or by fragmentation induced by collisions with a target gas or surface, or by photodissociation. Ions resulting from the fragmentation of molecular ions in the flight path can be observed at times given by the following formula:t=[(m/z)/2 eV]1/2 [L1+L2+4(m′/m)d]  (III)where m′ is the mass of the new fragment ion. In the case of peptides, these ions can provide amino acid sequences. These ions are generally focused by stepping or scanning the reflectron voltage VR or by using non-linear reflectrons (Cornish, T. J., Cotter, R. J., Non-linear Field Reflectron, U.S. Pat. No. 5,464,985, the entire contents of which is hereby incorporated by reference).
Other types of mass spectrometers include quadrupole mass spectrometers or quadrupole ion trap mass spectrometers. Quadupole ion trap mass spectrometers were first commercialized as detectors for gas chromatography (GC). In these first configurations developed by Finnigan Corporation, the ion trap mass spectrometer 40, shown schematically in FIG. 3, comprises electron impact ion source 42. Gaseous ions were produced from the GC effluent by electron impact ionization using electron source 42 (comprising a filament) and a fundamental radio-frequency 1.1 MHz was imposed on the ring electrode 44. With the end caps 46, 47 held at ground potential, mass spectra were recorded by scanning the amplitude of the fundamental RF frequency, causing the ions to fall out of the trapping field and into detector 48. This method is known as the mass-selective instability mode (Stafford, G. C., Jr., Kelley, P. E., Syka, J. E. P, Reynolds, W. E., Todd, J. F. J., Inter. J. Mass Spectrom. Ion Processes 60 (1984 85–98). However, this method of mass recording has a range of around 750 Da.
The addition of a supplemental radio-frequency (RF) voltage on the end cap electrodes enables resonant ejection of the ions while scanning the fundamental RF amplitude (Louris, J. N., Cooks, R. G., Syka, J. B. P., Kelley, P. E., Stafford, G. C., Jr., Todd, J. F. J., Analytical Chemistry 59 (1987) 1677–1685), This greatly increases the trap's mass range (from 2000 to 4000 Daltons on commercial instruments). In addition to their role in recording mass spectra, high amplitude supplemental waveforms can be used to isolate a specific preselected mass by ejection of all other ions (Louris, J. N., Brodbelt-Lustig, J. S., Cooks, R. G., Glish, G. L., Van Berkel, G. J., McLuckey, S. A., Ion Isolation and Sequential Stages of Mass Spectrometry in a Quadrupole Ion Trap Mass Spectrometer, Int. J. Mass Spect. Ion Processes 92 (1990) 117–137), while lower amplitude excitation can be used to provide repetitive low energy collisions that lead to fragmentation of the precursor mass (Practical Aspects of Ion Trap Mass Spectrometry: Fundamentals of Ion Trap Mass Spectrometry, March, Raymond E., Todd, J. F. J. (Eds.), CRC Press, Boca Raton (1995)). These and other types of excitation can employ both symmetric and non-symmetric waveforms and/or can be generated by stored waveform inverse Fourier transform SWIFT techniques (Soni, M. H., Cooks, R. G., Selective Injection and Isolation of Ions in Quadrupole Ion Trap Mass Spectrometry Using Notched Waveforms Created Using the Inverse Fourier Transform, Anal. Chem. 66 (1994) 2488–2496, and Doroshenko et al. U.S. Pat. No. 5,696,376). They form the basis of an ion trap's ability to perform MS/MS and MSn measurements.
The increased mass range capability led to the development of instruments with electrospray (Huang, P., Wall, D. B., Parus, S., Lubman, D. M., On-line Capillary Liquid Chromatography Tandem Mass Spectrometry on an Ion Trap/Reflectron Time-of-Flight Mass Spectrometer Using the Sequence Tag Database Search Approach for Peptide Sequencing and Protein Identification, J. Am. Soc. Mass Spectrom. 11 (2000) 127–135) and Matrix-Assisted Laser Desorption/Ionization (MALDI) (Doroshenko, V. M., Cornish, T. J., Cotter, R. J., Matrix-Assisted Laser Desorption/Ionization of Biological Molecules in the Quadrupole Ion Trap Mass Spectrometer, Anal. Chem. 65 (1993) 14–20). In these instruments, ions are formed outside the trap and various methods, including collisional cooling by an inert gas such as helium and dynamic gating of the fundamental trapping field (Doroshenko, V. M., Cotter, R. J., U.S. Pat. No. 5,399,857) were used to capture ions injected into the trap.
In the matrix-assisted laser desorption/ionization (MALDI) method, biomolecules to be analyzed are recrystallized in a solid matrix of a low mass chromophore. Following absorption of the laser radiation by the matrix, ionization of the analyte molecules occurs as a result of desorption and subsequent charge exchange processes. An ion trap mass spectrometer with a MALDI ion source is illustrated in FIG. 4. An ion trap mass spectrometer with MALDI 50 includes sample plate 52 and pulsed laser radiation 54 which is used to desorb and ionize the molecules under study. The ionic molecules are trapped and mass analyzed by ion trap mass spectrometer 56. Unlike TOF spectrometers, the mass measurement is not particularly sensitive to the ion's initial kinetic energy. The sample plate 52 is biased at a voltage sufficient to move the ions into the trap. The biasing voltage is generally considerably lower than the voltage used in TOF instruments. In the ion trap mass spectrometer/MALDI 50, pulsed extraction is not required for mass resolution since resonant frequency, not time, is the basis for mass ejection into detector 58.
The ion trap mass spectrometer-MALDI 50 allows obtaining MS/MS spectra for peptide molecular ions formed by MALDI. An MS/MS spectrum of an ovalbumin tryptic fragment is shown in FIG. 5. Although not all of the possible ions required for amino acid sequencing are observed, the eight ions indicated in FIG. 5 provide sequence tags that could be utilized in the identification of the peptide and its species of origin from a database.
Though ion traps are relatively small size instruments, interest in miniaturization of these mass analyzers has led to the development of cylindrical geometries (Badman, E. R., Cooks, R. G., Cylindrical Ion Trap Array with Mass Selection by Variation in Trap Dimensions, Anal. Chem. 72 (2000) 5079–5086 and Kornienko, O., Reilly, P. T. A., Whitten, W. B., Ramsey, J. M., Micro Ion Trap Mass Spectrometry, Rapid Commun. Mass Spectrom. 13 (1999) 50–53). A simplified geometry 60 shown in FIG. 6 makes it possible to construct multiple mass analyzers machined into a single assembly constructed from three parallel stainless steel plates 62, 63 and 64. The plates 62 and 64 play the role of end cap electrodes while the plate 63 plays the role of the radio-frequency ring electrode.
Hybrid instruments have also been developed using both ion trap and time-of-flight mass analyzers in tandem (He, L., Liu, Y.-H., Zhu, Y. Lubman, D. M., Detection of Oligonucleotides by External Injection into an Ion Trap Storage/Reflectron Time-of-Flight Mass Spectrometer, Rapid Commun. Mass Spectrom. 11 (1997) 1440–1448 and Doroshenko, V. M., Cotter, R. J., A Quadrupole Ion Trap/Time of-Flight Mass Spectrometer with a Parabolic Reflectron, J. Mass Spectrom. 33 (1998) 305–318). An example of such hybrid instruments is shown in FIG. 7. Hybrid mass spectrometer 70 comprises ion trap 72 and time-of-flight mass analyzer 74. The ion trap 72 is the first mass analyzer, which is used to trap ions, select a precursor, and induce fragmentation through the application of supplemental RF and collisions with a background gas (e.g., helium). The product ions are then mass analyzed by time-of-flight with TOF mass spectrometer 74. R. M. Jordan, Kratos Analytical and Syagen have commercialized ion trap/TOF tandem instruments. These instruments are capable of carrying out MSn cycles, where the last cycle is always recorded by a time-of-flight mass measurement. In these instruments, however, the mass range is determined by the highest mass that can be trapped in the ion trap 72 which may be from 2000 to 20000 Da.